Encapsulated adipose-derived stem cells, methods for preparation and theraputic use

ABSTRACT

A therapeutic composition comprising a purified fraction of adipose-derived mesenchymal stem cells encapsulated in a three-dimensional biocompatible gel matrix, and methods, and systems for preparing and using encapsulated adipose-derived mesenchymal stem cells. Hydrogel microbeads encapsulating stem cells maintain the viability and location of the stem cells for an extended period as compared to stem cells in suspension. The gel matrix allows the release of cellular factors from the encapsulated stem cells to surrounding tissues to achieve desired therapeutic results.

CLAIM OF PRIORITY

This application claims priority to U.S. Provisional Patent Application61/368,513, entitled “Methods And Compositions Using Therapeutic StemCells” filed Jul. 28, 2010; U.S. Provisional Patent Application61/368,528, entitled “Production Of Therapeutic Cells” filed Jul. 28,2010, both of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to therapeutic compositions includingstem cells derived from adipose tissue, including systems for preparing,delivering and utilizing such therapeutic compositions.

BACKGROUND OF THE INVENTION

Regenerative medicine can be defined as harnessing the body'sregenerative mechanisms in a clinically targeted manner, using them inways that are not part of the normal healing mechanism or byartificially amplifying normal mechanisms. Stem cells are pluripotent ormultipotent cells with the potential to differentiate into a variety ofother cell types, which perform one or more specific functions and havethe ability to self-renew. It has been found that stem cells from avariety of sources can be used for multiple therapeutic or prophylacticpurposes. For example, hematopoetic stem cells (HSCs) derived from bonemarrow are multipotent stem cells that can give rise to cell types fromthe myeloid (monocytes and macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells),and lymphoid lineages (T-cells, B-cells, NK-cells). Mesenchymal stemcells (“MSCs”) derived from multiple tissues in the adult body aremultipotent non-hematopoietic stem cells and are characterized byextensive proliferative ability in an uncommitted state while retainingthe potential to give rise to cell types including osteoblasts,myocytes, chondrocytes, adipocytes, endothelial cells and betapancreatic islet cells. MSCs are present in which arise from theembryonic mesoderm (e.g., hematopoietic cells and connective tissue).Thus, stem cells can be isolated from many tissue sources within theadult body.

Adipose tissue refers to fat including the connective tissue that storesthe fat. Adipose tissue includes stem cells and endothelial precursorcells. As used herein, “adipose tissue” refers to a tissue containingmultiple cell types including adipocytes and microvascular cells. It hasbeen discovered that adipose tissue is an especially rich and practicalsource of mesenchymal stem cells. This finding is due, at least in part,to the ease of harvesting adipose tissue and the ease of removing themajor non-stem cell component of adipose tissue, the adipocyte. In fact,a large quantity of mesenchymal stem cells can be obtained by simpleaspiration from adipose tissue, for example, from lipoaspirate samplesfrom aesthetic interventions. The lipoaspirate is typically centrifugedto separate the active cellular component from the mature adipocytes andconnective tissue. The pellet containing the active cellular component(e.g., the component containing adipose-derived stem cells) is referredto as processed lipoaspirate (PLA).

Adipose-derived stem cells (ADSCs), methods for extracting such cellsand methods for using such cells are disclosed for example in: Gimble etal., “Adipose-derived Stem Cells for Regenerative Medicine” Circ. Res.100:1249-1260 (2007); Utsonomiya et al., “Human Adipose-Derived StemCells: Potential Clinical Applications in Surgery” Surg Today 41:18-23(2011); Casteilla et al., “Adipose-derived stromal cells: Their identityand uses in clinical trials, an update” World J Stem Cells 3(4):25-33(2011); U.S. Pat. No. 6,777,231 entitled “Adipose-Derived Stem Cells andLattices” to Katz et al.; U.S. Pat. No. 7,901,672 entitled “Methods OfMaking Enhanced Autologous Fat Grafts” to Fraser et al.; U.S. PatentPublication 2009/0304644 entitled “Systems And Methods For ManipulationOf Regenerative Cells Separated And Concentrated From Adipose Tissue” toHedrick et al.; and U.S. Pat. No. 7,390,484 entitled “Self-ContainedAdipose Derived Stem Cell Processing Unit” to Fraser et al., all ofwhich are incorporated herein by reference.

SUMMARY OF THE INVENTION

Adipose-derived stem cells may be used for therapeutic and cosmeticapplications. Among other things, the cells may be used for regenerativemedicine, such as diseases that can be treated with regenerating cells.The present invention relates generally to therapeutic compositionsincluding adipose-derived stem cells derived from adipose tissue, aswell as systems for preparing, delivering and utilizing such therapeuticcompositions. The adipose-derived stem cells may be administered to apatient as part of a therapeutic composition as described herein. In apreferred embodiment, the adipose-derived stem cells are provided in athree-dimensional platform. The three-dimensional platform includesadipose-derived stem cells encapsulated in a biocompatible hydrogel andformed into microbeads which protect and support the stem cells whenintroduced to the human body thereby enhancing therapeutic efficacy ofthe treatment as compared to stem cells in suspension

These and other objects, features and advantages of the invention willbe apparent from the detailed description which follows.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the presently preferredembodiments of the invention. Although the disclosure herein refers tocertain illustrated embodiments, it is to be understood that theseembodiments are presented by way of example and not by way oflimitation. The intent of the following detailed description, althoughdiscussing exemplary embodiments, is to be construed to cover allmodifications, alternatives, and equivalents of the embodiments as mayfall within the spirit and scope of the invention as defined by theappended claims. The present invention may be practiced in conjunctionwith various cell or tissue separation techniques that areconventionally used in the art, and only so much of the commonlypracticed process steps are included herein as are necessary to providean understanding of the present invention.

Extraction and Isolation of Stem Cells

The present disclosure provides for the isolation and utilization ofstem cells. Such cells can be isolated from almost any embryonic oradult tissue including, but not limited to endothelial tissue fromumbilical cord vein, endothelial tissue from foreskin, endometrialtissue, human embryonic stem cells, and adipose tissue. Pluripotentcells can also be artificially produced by inducing pluripotency such asdescribed in Takahashi, K. & Yamanaka, S. Cell; 126: 663-676 (2006).

Mesenchymal Stem Cells (MSCs) are stem cells that can differentiatereadily into lineages including osteoblasts, myocytes, chondrocytes,adipocytes, endothelial cells and beta pancreatic islet cells(Pittenger, et al., Science, Vol. 284, pg. 143 (1999); Haynesworth, etal., Bone, Vol. 13, pg. 69 (1992); Prockop, Science, Vol. 276, pg. 71(1997)). MSCs, also known in the literature as bone marrow stem cells,skeletal stem cells, and multipotent mesenchymal stromal cells, arenon-hematopoietic progenitor cells isolated from adult tissues, and arecharacterized in vitro by their extensive proliferative ability in anuncommitted state while retaining the potential to differentiate alongvarious lineages of mesenchymal origin, including chondrocyte,osteoblast, and adipocyte lineages, in response to appropriate stimuli.In vitro studies have demonstrated the capability of MSCs todifferentiate into muscle (Wakitani, et al., Muscle Nerve, Vol. 18, pg.1417 (1995)), neuronal-like precursors (Woodbury, et al., J. Neurosci.Res., Vol. 69, pg. 908 (2002); Sanchez-Ramos, et al., Exp. Neurol., Vol.171, pg. 109 (2001)), cardiomyocytes (Toma, et al., Circulation, Vol.105, pg. 93 (2002); Fakuda, Artif. Organs, Vol. 25, pg. 187 (2001)) andpossibly other cell types. MSCs are present in multiple tissues in thebody which arise from the embryonic mesoderm (e.g., hematopoietic cellsand connective tissue). As such, pluripotent cells useful for thepresent invention can be isolated from any of these tissue sources andcan be induced to differentiate into any of these cell types.

In general, pluripotent cells are obtained from non-pathologicalpost-natal mammalian adipose tissues. Pluripotent cells can be obtainedfrom a source of adipose tissue, such as the stromal fraction of adiposetissue. The pluripotent cells can be obtained from any suitable sourceof adipose tissue from any suitable animal, including humans, havingadipose tissue. For example, adipose tissue can be obtained byconventional techniques known for the skilled person in the art (e.g.,liposuction), from any suitable source of adipose tissue from anysuitable animal, including mammals such as dogs, cats, horses, pigs,cows and humans. Preferably, pluripotent stem cells utilized in thepresent invention are derived from a mammal, such as from a human.

A convenient source of adipose tissue is from liposuction surgery. Infact, a large quantity of pluripotent cells can be obtained by simpleaspiration from adipose tissue, for example, from lipoaspirate samplesfrom aesthetic interventions. Because approximately 400,000 liposuctionprocedures are performed annually in the United States, this source ofpluripotent cells, particularly “mesenchymal stem cells” (MSCs) isparticularly promising for practicing the inventions disclosed herein.

In one instance, pluripotent cells of the invention are isolated fromadipose tissue. The adipose tissue can be obtained from an animal,preferably a mammal by any suitable method. A first step in any suchmethod requires the isolation of the adipose tissue from the sourceanimal. The animal can be alive or dead, so long as adipose stromalcells within the animal are viable. Typically, human adipose tissue isobtained from a living donor, using well-recognized protocols such assurgical or suction lipectomy. The preferred method to obtain humanadipose tissue is by excision or liposuction procedures well known inthe art. The pluripotent cells of the invention are present in theinitially excised or extracted adipose tissue, regardless of the methodby which the adipose tissue is obtained.

From whatever source, the tissue source containing pluripotent cells isprocessed to separate the pluripotent cells of the invention from theremainder of the tissue. Pluripotent cells can be obtained by washingthe tissue with a physiologically-compatible solution, such as phosphatebuffer saline (PBS). Typically, a washing step consists of rinsing theadipose tissue with PBS, agitating the tissue, and allowing the tissueto settle. In addition to washing, the adipose tissue can bedissociated. Dissociation can occur by enzyme degradation (e.g., trypsintreatment). Alternatively, or in conjunction with such enzymatictreatment, other dissociation methods can be used such as mechanicalagitation, sonic energy, or thermal energy. Cells are then centrifugedand the pellet (containing the pluripotent cells) is further treatedafter resuspension in an appropriate solution (e.g., PBS).

Pluripotent cells in the resuspended pellet can be separated from othercells of the resuspended pellet by methods that include, but are notlimited to, cell sorting, size fractionation, granularity, density,molecularly, morphologically, and immunohistologically (e.g., bypanning, using magnetic beads, FACS, MACS, or affinity chromatography.

In some immunologically-based methods of cell isolation, a pluripotentcell is obtained by positive selection, via the use of an antibody orother specific-binding protein, which binds to an epitope on the cellsurface. For example, a sample comprising cells from adipose tissue fromthree genetically distinct subjects is contacted with an antibody thatbinds to a surface molecule (e.g., CD105, CD44, TERT or CD 29) so as toform a cell-antibody-complex, recovering the pluripotent cells; therebyobtaining isolated, non-culture expanded pluripotent cells. Upon formingan antigen-antibody complex, the antigen-antibody complexes areseparated from the other subpopulations which are not bound to theantibody by a column, for example. Where the antibody is supported by amagnetic bead, a magnetic column can be used. The step of recovering thecells from the antibodies is performed by washes with suitable buffers,known to one skilled in the art. Alternately, pluripotent cells can beisolated by negative selection.

The presence of pluripotent cells may be verified by specific cellsurface markers which are identified with unique monoclonal antibodies,for example, see U.S. Pat. No. 5,486,359. Pluripotent cells of theinvention can proliferate and be induced to differentiate into cells ofother lineages by conventional methods. Methods for identifying andsubsequently isolating differentiated cells from their undifferentiatedcounterparts can be also carried out by methods well known in the art.See, e.g., Zheng et al. Rheumatology, 47:22-30 (2008). The capacity ofthe cells of the pluripotent cells of the invention to differentiateinto one or more cell lineages can be assayed by conventional methodsknown by the skilled person in the art.

Pluripotent cells of the invention are also capable of being expanded invitro. That is, after isolation, said cells can be maintained andallowed to proliferate in culture medium. Such medium is composed of anysuitable cell medium, for example, Dulbecco's Modified Eagle's Medium(DMEM), with or without antibiotics, and glutamine, and/or supplementedwith fetal bovine serum (FBS). It is within the skill of one in the artto modify or modulate concentrations of media and/or media supplementsas necessary for the cells used. Sera often contain cellular andnon-cellular factors and components that are necessary for viability andexpansion. Examples of sera include FBS, bovine serum (BS), calf serum(CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS),horse serum (HS), porcine serum, donkey serum, sheep serum, rabbitserum, rat serum (RS), etc. Where cells are of human origin,supplementation of cell culture medium with a human serum, for exampleof autologous origin, can be used. Modulation of serum concentrations,withdrawal of serum from the culture medium can also be used to promotesurvival of one or more desired cell types. In another embodiment,pluripotent cells of the invention can be expanded in a culture mediumof definite composition, in which the serum is replaced by a combinationof serum albumin, serum transferrin, selenium, and recombinant proteinsincluding but not limited to: insulin, platelet-derived growth factor(PDGF), and basic fibroblast growth factor (bFGF), as known in the art.

Many cell culture media already contain amino acids; however somerequire supplementation prior to culturing cells. Such amino acidsinclude, but are not limited to, L-alanine, L-arginine, L-aspartic acid,L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine,L-glycine, and the like. Antimicrobial agents are also typically used incell culture to mitigate bacterial, mycoplasmal, and fungalcontamination. Typically, antibiotics or anti-mycotic compounds used aremixtures of penicillin/streptomycin, but can also include, but are notlimited to amphotericin, ampicillin, gentamicin, bleomycin, hygromycin,kanamycin, mitomycin, etc. Hormones can also be advantageously used incell culture and include, but are not limited to, D-aldosterone,diethylstilbestrol (DES), dexamethasone, b-estradiol, hydrocortisone,insulin, prolactin, progesterone, somatostatin/human growth hormone(HGH), etc.

The maintenance conditions of pluripotent cell population(s) of theinvention can also contain cellular factors that allow cells to remainin an undifferentiated form. It is apparent to those skilled in the artthat prior to differentiation supplements that inhibit celldifferentiation must be removed from the culture medium. It is alsoapparent that not all cells require these factors and such factors maybe incompatible with maintaining the pluripotent phenotype of the cells.

If desired, pluripotent cell population(s) can be clonally expandedusing any suitable method for cloning cell populations. For example, aproliferated population of cells can be physically picked and seededinto a separate plate (or the well of a multi-well plate).Alternatively, the cells can be subcloned onto a multi-well plate at astatistical ratio for facilitating placing a single cell into each well(e.g., from about 0.1 to about 1 cell/well or even about 0.25 to about0.5 cells/well, such as 0.5 cells/well). The cells can be cloned byplating them at low density (e.g., in a Petri dish or other suitablesubstrate) and isolating them from other cells using devices such as acloning rings. The production of a clonal population can be expanded inany suitable culture medium. In any event, the isolated cells can becultured to a suitable point when their developmental phenotype can beassessed. In vitro expansion of pluripotent cells without inducingdifferentiation can be accomplished for example by using speciallyscreened lots of suitable serum (such as fetal bovine serum or humanserum).

Any of the steps and procedures for isolating pluripotent cells of theinvention can be performed manually, for example by microscopicevaluation based on phenotype and/or marker expression. Alternatively,the process of isolating such cells can be facilitated and/or automatedthrough one or more methods known in the art, for example, FACS or MACSsorting. For sorting and/or immunostaining of cells, an exemplary set ofmarkers are shown in Table 1. Cells may be positive for one, two, three,four, five, six, seven, eight, nine, ten or more markers of interest.One of skill in the art will recognize that such markers are providedonly as illustrations are not an exhaustive or limiting list of suchmarkers or tissue/cell types. Additionally, pluripotent cells may beidentified (e.g., by negative selection) for the absence of markers,including, but not limited to CD3, CD11b, CD14, CD19, CD31, CD34, CD45,CD62L, and HLA-DR.

TABLE 1 Non-limiting exemplary markers of select populations ofpluripotent cells Cell Source Markers Adipose tissue/umbilical cordCD13, CD29, CD44, CD49e, perivascular cells CD 54, CD71, CD73, CD90,CD105, CD95L, CD105, CD117, CD166, SOX2, TERT Endothelial tissue fromCD34, CD36, CD105, umbilical cord vein/foreskin CD150, CD151, CD160Endometrial cells SOX2, TERT, CD29, CD105, CD117 Chondrocyte-like cellsCollagen II, collagen X, aggrecan, ABCB1 (P-glycoprotein)

Also provided herein are methods of doing business in which cells of thepresent invention are collected and provided. Tissues can be collectedthrough any appropriate means, for example adipose tissue, foreskin andumbilical cord tissues can be collected by surgical or followingbirthing at a hospital or clinic. Tissues can be collected from multiplesites, including hospitals, clinics, physician's offices and tissuerepositories. Thus, tissues from multiple genetically distinctindividuals can be collected from the same source (e.g., hospital) aswell as multiple different sources. In some instances, tissues which arethe source of pluripotent cells can be transported to a central facilityfor combining, processing and/or extraction of pluripotent cells by anymethod known in the art, such as those described herein.

Methods for Extracting and Processing Adipose Tissue

In practicing the methods disclosed herein, the cells that areadministered to a patient are obtained from adipose tissue. Adiposetissue can be obtained by any method known to a person of ordinary skillin the art. For example, adipose tissue may be removed from a patient bysuction-assisted lipoplasty, ultrasound-assisted lipoplasty, andexcisional lipectomy. In addition, the procedures may include acombination of such procedures, such as a combination of excisionallipectomy and suction-assisted lipoplasty. As the tissue or somefraction thereof is intended for reimplantation into a patient theadipose tissue should be collected in a manner that preserves theviability of the cellular component and that minimizes the likelihood ofcontamination of the tissue with potentially infectious organisms, suchas bacteria and/or viruses. Thus, the tissue extraction should beperformed in a sterile or aseptic manner to minimize contamination.

For suction-assisted lipoplastic procedures, adipose tissue can becollected by insertion of a cannula into or near an adipose tissue depotpresent in the patient followed by aspiration of the adipose into asuction device. In one embodiment, a small cannula may be coupled to asyringe, and the adipose tissue may be aspirated using manual force.Using a syringe or other similar device may be desirable to harvestrelatively moderate amounts of adipose tissue (e.g., from 0.1 ml toseveral hundred milliliters of adipose tissue).

Suction assisted lipoplasty may be desirable to remove the adiposetissue from a patient as it provides a minimally invasive method ofcollecting tissue with minimal potential for stem cell damage. Asuitable system includes a single-use disposable aspiration system,which allows extraction of adipose into sterile sealed bag for furtherprocessing. Preferably the system employs a relatively small devicewhich has the advantage that the lipoaspiration can be performed withonly local anesthesia, as opposed to general anesthesia.

The adipose tissue that is removed from a patient is collected into asterile container for further processing. The device is designed for anddedicated to the purpose of collecting tissue for manufacture of aprocessed adipose tissue cell population, which includes stem cellsand/or endothelial precursor cells. In some cases, the device is asingle-use disposable aspiration system which extracts adipose tissuefrom a patient into a sterile sealed bag. In alternative embodiments,the device may be any conventional device that is typically used fortissue collection by physicians performing the extraction procedure.

The amount of tissue collected will be dependent on a number ofvariables including, but not limited to, the body mass index of thedonor, the availability of accessible adipose tissue harvest sites,concomitant and pre-existing medications and conditions (such asanticoagulant therapy), and the clinical purpose for which the tissue isbeing collected. Moreover, the stem cell percentage of 100 ml of adiposetissue varies from individual to individual. For example theconcentration of stem cells in tissue extracted from a lean individualis greater than that extracted from an obese donor). This reflects adilutive effect of the increased fat content in the obese individual.During preparation of therapeutic compositions including the stem cellsit is advantageous to quantify and characterize the extracted stem cellpopulation in order to control the number of stem cells incorporatedinto the therapeutic composition and delivered to the patient.

The preferred method to obtain human adipose tissue is by excision orliposuction procedures well known in the art. The pluripotent cells ofthe invention are present in the initially excised or extracted adiposetissue, regardless of the method by which the adipose tissue isobtained. The adipose tissue is then processed to facilitateseparation/concentration of the stem cells. For example, pluripotentcells can be obtained by washing the tissue with aphysiologically-compatible solution, such as phosphate buffer saline(PBS). Typically, a washing step consists of rinsing the adipose tissuewith PBS, agitating the tissue, and allowing the tissue to settle. Inaddition to washing, the adipose tissue can be dissociated. Dissociationcan occur by enzyme degradation (e.g., trypsin treatment).Alternatively, or in conjunction with such enzymatic treatment, otherdissociation methods can be used such as mechanical agitation, sonicenergy, or thermal energy. Cells are then centrifuged and the pellet(containing the pluripotent cells) is further treated after resuspensionin an appropriate solution (e.g., PBS).

Pluripotent cells in the resuspended pellet can be separated from othercells of the resuspended pellet by methods that include, but are notlimited to, cell sorting, size fractionation, granularity, density,molecularly, morphologically, and immuno-histologically (e.g., bypanning, using magnetic beads, FACS, MACS, or affinity chromatography.In some immunologically-based methods of cell isolation, a pluripotentcell is obtained by positive selection, via the use of an antibody orother specific-binding protein, which binds to an epitope on the cellsurface. The step of recovering the cells from the antibodies isperformed by washes with suitable buffers, known to one skilled in theart. Alternately, pluripotent cells can be isolated by negativeselection. In the present invention, the kit includes sterile containersand/or reagents to facilitate the separation/concentration of the stemcells in the lipoaspirate. The presence of pluripotent cells ispreferably assessed prior to reintroduction using, for example, specificcell surface markers and/or counting techniques. Such assessment allowsquantification and characterization of the extracted stem cellpopulation in order to control the number of stem cells incorporatedinto a therapeutic composition and delivered to the patient.

Phenotypic Alteration/Differentiation of Stem Cells

Pluripotent cells obtained using the system of the present invention canbe reintroduced to the patient without expansion. However, inalternative embodiments, the pluripotent cells are optionally expandedin vitro. That is, after isolation, said cells can be maintained andallowed to proliferate in culture medium. It is within the skill of onein the art to modify or modulate concentrations of media and/or mediasupplements as necessary for the cells used. In another embodiment,pluripotent cells of the invention can be expanded in a culture mediumof definite composition, in which the serum is replaced by a combinationof serum albumin, serum transferrin, selenium, and recombinant proteinsincluding but not limited to: insulin, platelet-derived growth factor(PDGF), and basic fibroblast growth factor (bFGF), as known in the art.The maintenance conditions of pluripotent cell population(s) of theinvention can also contain cellular factors that allow cells to remainin an undifferentiated form where undifferentiated stem cells aredesired.

Pluripotent cells obtained using the system of the present invention canbe reintroduced to the patient without phenotypicalteration/differentiation. The pluripotent cells can alternatively beinduced to phenotypically change into a desired cell type. Pluripotentcells can be directed to phenotypically change/differentiate into adesired cell type, e.g., a chondrocyte, or a cell with properties of adesired cell type, e.g., a chondrocyte-like cell (for example, a cellexpressing collagen II, collagen X, aggrecan, ABCB1, or a combination ofthese). Such phenotypic changes are specific to the cell type to beproduced as are the methods for directing differentiation.

Typically, one or more additional substances can be added to the cultureto induce a desired phenotypic change. Such substances can includeactivators of intracellular signaling pathways (mitogen-activatedprotein kinases and Smads), activators of transcription factors (sox9,L-sox5, and L-sox6), activators of production and interaction withextracellular matrix proteins (collagen II, aggrecan, and cartilageoligomeric matrix protein), growth factors, cytokines, chemokines,hormones and environmental factors (oxygen tension). Non-limitingexamples of substances which can induce a change to a chondrocyte-likecell include, but are not limited to dexamethasone,beta-glycerophosphate, ascorbic acid, transforming growth factor, and/orbis(bromomethyl)propanediol (BMP).

MSCs can also be cultured in a three-dimensional matrix to induce adesired phenotypic change. For example, production of chondrocyte-likecells from MSCs or other pluripotent cells can comprise athree-dimensional culture of MSCs utilizing a three-dimensional scaffoldcomprised of extracellular matrix. Multiple types of matrices can beused to support the pluripotent cells as they differentiate. One form ofmatrix is a polymeric mesh or sponge; another is a polymeric hydrogel.Typically, a matrix is biodegradable over a time period of less than ayear. Such extracellular matrices can be composed of any suitablematerial, including collagen, albumin, and fibrin; and polysaccharidessuch as alginate and polymers of hyaluronic acid, polylactic acid (PLA),polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA),polyorthoesters, polyanhydrides, polyphosphazenes, and combinationsthereof.

Alternately, pluripotent cells can be cultured in a “micromass” culturecomprising artificially conglomerated cells (e.g., by centrifuging acell suspension to produce a pellet) which is substantially free ofextracellular matrix. As used herein, “substantially free ofextracellular matrix” means that a composition contains only anegligible amount of any exogenously added extracellular matrix, but maycontain such materials produced by the cells.

Therapeutic Compositions of Stem Cells

For the administration in the prevention and/or treatment of a disorder,such as joint disorders (e.g., osteoarthritis), cells of the invention(e.g., chondrocyte-like cells derived from pluripotent cells) can beformulated in a suitable pharmaceutical composition, comprising cells ofthe invention, in a therapeutically or prophylactically effectiveamount, together with a suitable pharmaceutically acceptable vehicle.The pharmaceutical composition of the invention can contain aprophylactically or therapeutically effective amount of the cells of theinvention, preferably in a substantially purified form, together withthe suitable vehicle in the appropriate amount in order to provide theform for proper administration to the subject. One form of therapeuticcomposition includes stem cells encapsulated in hydrogel as part of a3-dimensional platform as described below.

As used herein the term “prophylactically or therapeutically effectiveamount” refers to the amount of cells of the invention contained in thepharmaceutical composition which is capable of producing the desiredtherapeutic effect. One of skill in the art will recognize that cellnumbers (e.g., dosage amount) will vary depending upon multiple factorsincluding, but not limited to site of administration, extent of disease,and method of administration. For example, an administration directlyinto the joint of a subject suffering from OA will typically contain asmaller number of cells than an administration of the cells into thebloodstream. The dose of cells disclosed herein can be repeated,depending on the patient's condition and reaction, at time intervals ofdays, weeks or months as determined necessary by a treating physician orother healthcare professional. As previously described, assessment ofthe extracted stem cell population prior to incorporation into apharmaceutical composition allows quantification and characterization ofthe extracted stem cell population in order to control the number ofstem cells incorporated into a therapeutic composition therebyfacilitating delivery of a desired prophylactically or therapeuticallyeffective amount of stem cells to the patient.

The pharmaceutical composition of the invention can be formulatedaccording to the chosen form of administration. For example, apharmaceutical composition is prepared in a liquid dosage form, e.g., asa suspension, to be injected into the subject in need of treatment.Illustrative, non-limiting examples, include formulating thepharmaceutical composition of the invention in a sterile suspension witha pharmaceutically acceptable vehicle, such as saline solution,phosphate buffered saline solution (PBS), or any other suitablepharmaceutically acceptable carrier, for parenteral administration to asubject, e.g., a human being, preferably via intravenous,intraperitoneal, subcutaneous, etc., although further administrationroutes may be also possible.

If desired, the cells of the present invention can be purified, afterinduction of phenotypic alteration by use of antibody-mediated positiveand/or negative cell selection in order to enrich the cell population.Phenotypically altered cells of the invention (e.g., chondrocyte-likecells) can be administered to a subject without further processing oradditional procedures to further purify, modify, stimulate, or otherwisechange the cells. Alternately, phenotypically altered cells can bepurified (e.g., by positive selection) such that a compositioncomprising the cells (e.g., chondrocyte-like cells) is substantiallyfree of pluripotent cells.

The term “pharmaceutically acceptable vehicle” refers to a compositionapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia, or European Pharmacopeia, or othergenerally recognized pharmacopeia for use in animals, including humans.The term “vehicle” refers to a diluent, adjuvant, excipient, or carrierwith which the cells of the invention are administered, thus, thevehicle must be compatible with the cells. Examples of suitablepharmaceutical vehicles are described in “Remington's PharmaceuticalSciences” by E. W. Martin. Illustrative, non-limiting, examples ofvehicles for the administration of cells contained in a pharmaceuticalcomposition of the invention include, for example, a sterile salinesolution (0.9% NaCl), PBS, etc.

The pharmaceutical compositions of the invention, if desired, can alsocontain, when necessary, additives to enhance, control, or otherwisedirect the intended therapeutic effect of the cells comprising saidpharmaceutical composition, and/or auxiliary substances orpharmaceutically acceptable substances, such as minor amounts of pHbuffering agents, tensioactives, co-solvents, preservatives, etc. Also,for stabilizing the cell suspension, it is possible to add metalchelating agents. The stability of the cells in the liquid medium of thepharmaceutical composition of the invention can be improved by means ofadding additional substances, such as, for example, amino acids such asaspartic acid, glutamic acid, etc. Pharmaceutically acceptablesubstances that can be used in the pharmaceutical composition of theinvention are known, in general, by the skilled person in the art andare normally used in the manufacture of cellular compositions.

In one example of a therapeutic composition, chondrocyte-like cells areproduced from isolated pluripotent cells by any of the methods describedherein. Chondrocyte-like cells are then prepared for application tosubjects in need of the cells. Chondrocyte-like cells can also beprepared in pharmaceutical dosages (e.g., in a pharmaceuticallyacceptable solution) and stored in appropriate containers. Thechondrocyte-like cells can be stored in an appropriate manner (e.g.,frozen) until needed. Additionally, the pharmaceutical dosages can beplaced in pre-prepared syringes, catheters or other medical devicesappropriate for delivery to an affected joint. One of skill in the artwill recognize that dosage amount, needle length and other suchparameters can be adjusted for any individual preparation.

A pharmaceutical composition containing cells of the present inventionmay be stored until use by means of conventional methods known by theskilled person in the art. For short term storage (less than 6 hours)the pharmaceutical composition containing said cells may be stored at orbelow room temperature in a sealed container with or withoutsupplementation with a nutrient solution. Medium term storage (less than48 hours) is preferably performed at 2-8° C., the pharmaceuticalcomposition comprising an iso-osmotic, buffered solution in a containercomposed of or coated with a material that prevents cell adhesion.Longer term storage is preferably performed by appropriatecryopreservation and storage under conditions that promote retention ofcellular function.

Cells in either prepared dosages or pre-dosage containers can be shippedto medical facilities through any approved delivery system(governmentally approved and/or commercial). Cells can be delivereddirectly from the manufacturer or via an intermediary. Fees can becollected for delivery of the cells through any appropriate means (e.g.,credit card, credit account, cash, check, etc.).

Encapsulation of Stem Cells—3D Platform

In an embodiment of the invention, a 3-dimensional (3D) platform iscreated to support cells introduced as a therapeutic composition intothe body. In general terms the platform is created by extraction of SVMwith or without purification of stem cells. The cells are dispersed in abiocompatible gel/polymeric matrix. Preferably the gel is biocompatibleand biodegradable, for example a fibrin hydrogel. The gel containing thestem cells is then formed into microbeads thereby increasing the activesurface area of the therapeutic composition. The formation of themicrobeads can be performed manually or mechanically. Preferably, themicrobeads are of 10 to 50 μL in size and each bead includes from 2,000to 10,000 stem cells. The concentration of stem cells in the microbeadsis selected so as to be high enough to achieve the desired therapeuticeffect without reduced the effectiveness of the encapsulation andviability of the stem cells. A suitable concentration of stem cells hasbeen found to be 200 stem cells per μL. This concentration of stem cellshas been found to promote maintenance and viability of the stem cellswithin the microbeads for up to 14 days. The therapeutic composition ofmicrobeads of gel containing stem cells can then be injected or appliedsuperficially as a therapeutic composition or to augment autologousadipose tissue transplant procedures.

The 3D platform provides significant advantages compared to a dispersedsuspension of stem cells. For example, the stem cells remain in groups,keeping on interaction, normal proliferation and gross factor secretionwhereas in suspension, the single stem cells are unable to sustainnormal development in form of single cells in suspension. Additionally,the 3D platform defends the encapsulated stem cells againstenvironmental changes and mechanical stress upon delivery of the stemcells into the hosting tissue. The 3D matrix supports the stem cellsassuring normal metabolism over an extended period as compared to asuspension of stem cells. The support of the stem cells by the 3Dplatform also enhances the storage or cryostorage of the stem cellsfacilitating maintenance, transport and delivery of stem cells at thetime and place required for a procedure. Additionally, the initialamount of available and injected cells can be determined duringpreparation of the 3D platform, which allows development ofdose-dependent controlled treatment. This is facilitated, as describedabove, by characterization of the stem cell population prior to and/orduring preparation of the therapeutic composition.

The advantages of the 3D platform provide significant therapeuticbenefits. The encapsulated stem cells in the 3D platform can be moreaccurately delivered to a precise location in the human body and willremain in the targeted location—as opposed to stem cells in suspensionwhich rapidly dissipate from the injection site. Moreover, as describedabove, the 3D platform increases the longevity and functionality of thestem cells by protecting them against chemical and mechanical stress atthe injection site. Maintaining the stem cells in the target locationand extending their viability extends the treatment effects of the stemcells thereby enhancing the treatment and or reducing the need forrepetition of the treatment.

In a preferred embodiment adipose-derived stem cells are encapsulated inthe 3D platform to generate a therapeutic composition. Preparation ofencapsulated stem cells requires three general steps. First, stem cellsmust be extracted, and isolated. The extraction and isolation of stemcells can be performed using conventional methods and/or the methodsdescribed herein. Second, the stem cells are mixed with a liquid phasebiocompatible pro-polymer. Third, the mixture is caused to gel bycrosslinking of the pro-polymer to form a polymer. As result, the stemcells are embedded in polymeric biodegradable hydrogel network whichserves as 3D culture and support system for the stem cells. A range ofbiocompatible polymers are know to those of skill in the art including,for example, fibrin, alginate and collagen polymers. A suitable polymeris biocompatible and biodegradable but provides suitable mechanical andchemical support to the stem cells during a period over which they canhave therapeutic effect. In a preferred embodiment, a Fibrin/thrombingel is used suitable for maintaining the stem cells for a period ofthree to fourteen days.

A therapeutic composition comprising encapsulated stem cells can beintroduced into tissues adjacent the site of an injury. Because thecells are encapsulated they do not migrate away from the site ofintroduction. However, the stem cells can proliferate in vivo. Moreover,the presence of the encapsulated stem cells can stimulategrowth/proliferation of tissues adjacent the encapsulated stem cells by,for example, the release of cytokines, growth factors andanti-inflammatory factors. It is thought that encapsulated stem cellsintroduced in this manner can achieve regenerative healing withoutdifferentiation and integration of the stem cells actually introduced.Indeed, in some embodiments, encapsulation of the stem cells extends theperiod of the treatment effect by maintaining the stem cells inundifferentiated form isolated from factors in the tissue which mightengender differentiation of the stem cells. Thus, in some embodiments itis desirable to introduce the stem cells adjacent the site of an injuryrather than directly at the site of an injury.

Administration of Therapeutic Compositions

The administration of the pharmaceutical composition of the invention tothe subject in need thereof can be carried out by conventional means. Ina particular embodiment, said pharmaceutical composition can beadministered to the subject in need by intravenous administration usingdevices such as syringes, catheters, trocars, cannulae, etc. In anycase, the pharmaceutical composition of the invention will beadministrated using the appropriate equipments, apparatus, and deviceswhich are known by the skilled person in art in a therapeutically orprophylactically effective amount, together with a suitablepharmaceutically acceptable vehicle.

Cells disclosed herein can be applied by several routes includingsystemic administration by venous or arterial infusion (includingretrograde flow infusion) or by direct injection into the affectedanatomical site. A pharmaceutical composition containing the cells maybe injected in a single bolus, through a slow infusion, or through astaggered series of applications separated by several hours, severaldays or weeks. In any case, the pharmaceutical composition of theinvention will be administrated to the target tissue using theappropriate equipments, apparatus, and devices which are known by theskilled person in art in a therapeutically or prophylactically effectiveamount. In alternative embodiments, the 3D platform can be utilized toaugment fat autografts implanted using conventional techniques.

One of skill in the art will recognize that cell numbers (e.g., dosageamount) will vary depending upon multiple factors including, but notlimited to site of administration, extent of disease, and method ofadministration. For example, an administration directly into the jointof a subject suffering from OA will typically contain a smaller numberof cells than an administration of the cells into the bloodstream. Thedose of cells disclosed herein can be repeated, depending on thepatient's condition and reaction, at time intervals of days, weeks ormonths as determined necessary by a treating physician or otherhealthcare professional.

In a preferred embodiment, stem cells are encapsulated as part of a 3Dplatform as described above. The 3D platform incorporating the stemcells are then delivered to a target location to achieve the desiredtherapeutic effect. The 3D platform can be administered to the subjectin need by direct administration into target tissue using devices suchas syringes, catheters, trocars, cannulae, etc. In a preferredembodiment the 3D platform is administered percutaneously under imageguidance to a desired location.

One mode of treatment introduces stem cells encapsulated in the 3Dplatform adjacent tissues to be treated. The 3D platform maintains thestem cells in their undifferentiated from and protects the stem cellsfrom chemical and mechanical stress at the site of introduction. Theencapsulated stem cells are able to survive and/or proliferate in vivofor an extended period as compared to stem cells in suspension. Theencapsulated stem cells release cytokines, growth factors,anti-inflammatory factors which migrate out of the 3D platform into thesurrounding tissues. These factors engender a therapeutic effect in thetarget tissues adjacent the site of injection of the 3D platform.

In one example, a 3D platform encapsulating stem cells is used to treatosteoarthritis. Rather than injecting a suspension of stem cellsdirectly into a joint, the 3D platform is introduced sub-chondrallyusing an image-guided needle or similar technology. The stem cells inthe 3D platform do not themselves differentiate into tissues to repairthe joint. However cellular factors released from the 3D platform areable to migrate into the osteo-arthritic tissues of the jointrejuvenating the tissues and thereby stimulate those tissues to repairthemselves. Essentially the 3D platform permits the release of factorsfrom the encapsulated stem cells with reduce inflammation and triggerself repair in the affected tissues of the joint such as the damagedcartilage. Triggering self-repair of the tissues allows for creation ofdefect repairs which more closely approximate the natural tissue. Thisis advantageous to direct injection of stem cells to differentiate intorepair tissue because such approach generally results in tissues thatdoe not have the desired structural features of the natural tissues.

Cells and pharmaceutical compositions of the present disclosure can beused in a combination therapy with other substances useful for treatingthe same disorder. Such combination therapy can comprise the cells ofthe present invention directly combined with other substances (e.g.,other pharmaceuticals), or in conjunction with other substances.Combination therapy can also include delivery of therapeuticcompositions as described herein along with

Autologous/Allogeneic Origin of Stem Cells

The cells disclosed herein can be cells of autologous or allogeneicorigin. Autologous stem cells are derived from the individual into whomthe stem cells are later reinfused. Autologous stem cells areadvantageous in that, being the individuals own tissue, they do notengender an immune response from the recipient. Allogeneic stem cells onthe other hand, are derived from one or more individuals other than therecipient. These stem cells can illicit an immune/rejection response insome circumstances. Steps can be taken to reduce the chance ofrejection, such as by using tissue-matched donors. Allogeneic stem cellscan still be utilized in certain applications. Although autologous stemcells are preferred, allogeneic stem cells can, for example be utilizedas part of a 3D-platform described above—the allogeneic stem cells areeffective to deliver therapeutic cellular factors for a period afterintroduction to the patient and are protected from rejection by the 3Dplatform at least for the limited period during which they have theirtherapeutic effect.

Where cells of allogeneic origin are to be used, for example, extractedadipose tissue from two or more genetically distinct individuals iscombined prior to isolation of pluripotent cells from the tissue. Theterm “genetically distinct” as used herein, indicates that at least onedifference at the genomic level exists between subjects or donors.Adipose tissue can be collected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100 or more subjects or donors. In some instances,cells for use in treating a subject (e.g., chondrocyte-like cells) arederived from pluripotent cells donated from two or more geneticallydiverse subjects. For example, MSCs from adipose tissue collected fromliposuction performed on different individuals can be pooled prior toculturing under conditions which lead to chondrocyte-like cellproduction. Alternately, cells from a single individual may be inducedto phenotypically switch to chondrocyte-like cells and then combined.Such an approach allows for immediate treatment of individuals sufferingfrom joint disorders such as osteoarthritis by using “off the shelf”cells. Without being bound by theory, such an approach can be utilizeddue to the relative lack of immune surveillance at the site of insertion(e.g., the synovial space). If desired, the cells of the presentinvention can be purified, after induction of phenotypic alteration byuse of antibody-mediated positive and/or negative cell selection inorder to enrich the cell population. Phenotypically altered cells of theinvention (e.g., chondrocyte-like cells) can be administered to asubject without further processing or additional procedures to furtherpurify, modify, stimulate, or otherwise change the cells. Alternately,phenotypically altered cells can be purified (e.g., by positiveselection) such that a composition comprising the cells (e.g.,chondrocyte-like cells) is substantially free of pluripotent cells.

Using the methods and compositions provided herein the presentdisclosure provides other methods which can be used to providetherapeutic cells (e.g., chondrocyte-like cells) to a plurality ofpatients. For instance, therapeutic cells can be produced in largebatches comprising cells derived from multiple genetically distinctdonors (e.g., from multiple liposuction patients). Liposuction (orlipectomy) is a common procedure in the United States, withapproximately 400,000 such procedures performed each year. Cells fromsuch procedures can be procured from multiple locations and multiplegenetically-distinct individuals. As described above, pluripotent cellsfrom such sources can be isolated to produce the therapeutic cellsdescribed herein in large numbers (e.g., millions, billions, trillions).

As therapeutic cells provided to an individual in need thereof can beallogeneic with respect to the recipient (e.g., patient suffering fromosteoporosis), the methods described herein provide therapeutic cellpopulations which can be produced on a large scale. In some instances,cells sufficient to treat 10, 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 5000, 10,000, 20,000, 50,000, 100,000, 500,000,1,000,000, 10,000,000, 50,000,000, 100,000,000 or more subjects can beproduced. Therapeutic cells can be produced in large quantities anddivided into smaller populations for distribution. Smaller divisions caninclude packaging into individual treatment packages (e.g., a pre-loadedsyringe), or other appropriate sized forms (e.g., canisters containingsufficient cells for multiple treatments). Therapeutic cells can beproduced prior to an individual patient's need (e.g., days, weeks, ormonths prior to such need) or can be produced in response to anindividual patient's need.

For example, a sample comprising cells from adipose tissue from severalgenetically distinct donors is combined with antibodies, which aredirected to the surface of other subpopulation(s) which exist in thesample (e.g., CD11b, CD14, CD19), thereby trapping the non-pluripotentcells. One of skill in the art will recognize that such selection ofcells can be performed prior to culturing cells, following cell culture,or both. Thus, in some embodiments, pluripotent cells from one or moregenetically distinct individuals may be substantially free ofnon-pluripotent cells. Furthermore, pluripotent cells from multipleindividuals can be collected and/or combined prior to culturing,following culturing, or both. Thus, pluripotent cells from 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more individuals canbe combined.

EXAMPLES Example 1 Isolation and Culture of Adipose Derived Pluripotent(Stem) Cells

In this example, pluripotent cells are isolated from human adiposetissue. Adipose tissue is collected from multiple anonymous donorsduring surgical procedures, such as liposuction. The tissue is mincedinto pieces ranging from 1-2 mm³, washed with phosphate-buffered saline(PBS) and digested with 1.5 mg collagenase type I per mg of adiposetissue for 60 minutes at 37° C. Cells are subjected to multiplecentrifugations and floating cells are removed. The cell mixture isexposed to erythrocyte lysis buffer, incubated at room temperature for10 minutes, and centrifuged at 300×g for 10 minutes. The supernatant isremoved. Collected cells are grown for 3-4 generations in DMEM+10% FBSat 37° C.

Approximately 10⁷ cells are transferred to a 15 mL polypropylene conicaltube, centrifuged at 300×g for 10 minutes and the supernatant isaspirated. To enrich the population for pluripotent cells the resultingpellet is resuspended in 80 μL PBS. FcR blocking buffer and anti-CD105antibody conjugated with allophycocyanin (APC) is added to the cellsuspension according to manufacturer's instructions. The suspension ismixed well and incubated at 2-8° C. for 10 minutes. One ml PBS is addedand cells are centrifuged at 300×g for 10 minutes and the supernatant isaspirated. Cells are resuspended in 70 μL PBS. 10 μL FcR blockingreagent and 10 μL Anti-APC MicroBeads (Miltenyi Biotec) are added to thesuspension. Cells are incubated at 2-8° C. for 15 minutes. Cells arewashed by adding 1 ml PBS and centrifuged at 300×g for 10 minutes andthe supernatant is aspirated. Cells are resuspended in 500 μl PBS. Cellsare then run through an autoMACS Column (Miltenyi Biotec) according tomanufacturer's instructions. CD105 positive cells are cultured inProliferation Medium for 3-5 days at 37° C.

Example 2 Harvesting and Differentiation of Pluripotent Cells

In this example, CD105-positive cells are harvested from culture anddifferentiated into chondrocyte-like cells. Cultured CD105-positivecells prepared as described in Example 1 are collected from culture andwashed by adding 2 ml PBS to the tissue culture flask and aspirating thesupernatant. One ml trypsin/EDTA (0.05%/0.53 mM) is added to cover thecells and the mixture is incubated at 37° C. for 5-10 minutes.Dissociation of cultured cells is examined under a microscope. Gentletapping of the flask or increased incubation time may be necessary tofacilitate further dissociation as needed. Five ml pre-warmedproliferation medium is added to the flask upon sufficient dissociationand the cells are resuspended by pipetting and transferred to a 15 mlpolypropylene conical tube. The flask is washed with an additional 5 mlproliferation medium, which is also transferred to the conical tube tocollect all cells. Cells are centrifuged at room temperature at 300 gfor 10 minutes. The supernatant is removed and the cells are gentlyresuspended in 0.5 ml proliferation medium. Cell number and viability isdetermined by exposing 10 μL cells to Trypan Blue and using ahemocytometer. Cells are then diluted to a final concentration of 3×10⁵cells/ml in the expansion medium.

To cause the CD105-positive cells to differentiate into chondrocyte-likecells, the following protocol is One ml of the cell suspension istransferred to a 15 ml polypropylene conical tube and centrifuged for 5minutes at 150 g at room temperature. The supernatant is aspirated and 1ml pre-warmed Differentiation Medium (20 ml DMEM+10% FBS with 2 mlgrowth factor cocktail (10 μl/ml beta-TGF1, 5 μl/mlbis(bromomethyl)propanediol, and 0.1 μl/ml dexamethasone). Cells arecentrifuged for 5 minutes at 150 g at room temperature and thesupernatant is aspirated. 1 ml pre-warmed Differentiation Medium isadded, but the cells are not suspended. The tube is incubated upright at37° C. with 5% CO2, 95% N2 and >95% humidity. Fresh DifferentiationMedium is added every third day, being careful not to disturb the cellpellet. After 28 days, the nodules (micromass culture) have matured andcan be frozen and stored or transferred to a recipient.

Example 3 Determination of Chondrocyte-Like Phenotype

In this example, cells prepared as detailed in Examples 1 and 2 areexamined to determine whether they have differentiated intochondrocyte-like cells. Formalin is diluted with PBS to a finalconcentration of 3.7% (neutral buffered formalin or “NBF”), ethanol isdiluted with deionized water to concentrations as needed. Roti-Plast(paraffin) is heated at 58° C. until melted.

Differentiation Medium is aspirated from a micromass culture prepared asdescribed in Example 2 and is washed once with 1 ml PBS. The micromassis fixed by immersion in NBF (the micromass or nodule is freelysuspended in NBF) for 6-12 hours at room temperature with gentleagitation. Nodules are placed in an embedding cassette with filter paperand dehydrated by applying ethanol as follows: 2×30 min in 70% ethanol,2×30 min in 80% ethanol, 2×30 min in 90% ethanol, and 2×30 min in 100%ethanol. The embedding cassette is then incubated in Roti-Histol (xylolsubstitute—Roth). Nodules are then removed from the embedding cassetteand embedded with preheated Roti-Plast in a Bio-mold and cooled at −20°C. overnight.

Five μm thick tissue sections are prepared using a microtome and thesections are transferred to a 40° C. water bath. Tissue sections areplaced on HistoBond slides, incubated at 52° C. for 3 hours and thencooled to room temperature before staining or storage. Sections to beanalyzed are deparaffinized using 2×5 min in Roti-Histol, 2×5 min in100% ethanol, 2×5 min in 96% ethanol, 2×5 min in 80% ethanol and 2×5 minin 70% ethanol. Sections are rinsed twice in deionized water and twicein 5 mm PBS. Sections are then incubated in permeabilization buffer (PBSwith 1% BSA, 10% normal donkey serum and 0.3% Triton X-100) for 45 minat room temperature. The permeabilized section is then dabbed dry andencircled using a hydrophobic pen. Mouse anti-human aggrecan antibody(10 μg/ml) in PBS with 1% BSA and 10% normal donkey serum is thenapplied (150 μL) and incubated overnight at 2-8° C. in a humidifiedchamber. Sections are washed with washing buffer (PBS+1% BSA) threetimes for five minutes. 150 μl donkey anti-mouse IgG conjugated withrhodamine (diluted 1:50 in washing buffer) is added and incubated atroom temperature for 1 hour in the dark. The secondary antibody isremoved by tilting the slide and gently removing by blotting. 150 μLDAPI (diluted 1:1000 in washing buffer) is added to each section andincubated in the dark for 15 minutes. Sections are then washed twicewith washing buffer in the dark and rinsed once with deionized water.Sections are examined using a fluorescent microscope for positivesignals indicating the presence of aggrecan, and thus a phenotypic shiftto chondrocyte-like cells.

Example 4 Treatment of Osteoarthritis by Injection of Chondrocyte-LikeCells

In this example, treatment of osteoarthritis is performed by injectingchondrocyte-like cells derived from adipose tissue pluripotent cells.Nodules of chondrocyte-like cells developed as described above aretreated with EDTA, EGTA or enzymatic digestion (e.g., trypsin) andresuspended. Cell suspensions are then injected into an affected knee orelbow joint.

The administration of the chondrocyte-like cells can be carried out byconventional means. In a particular embodiment, chondrocyte-like cellscan be administered into the joint of subject in need using devices suchas syringes, catheters, trocars, cannulae, etc. The administration canbe performed percutaneously/arthroscopically and can be repeated asnecessary to achieve the desired therapeutic effects. In any case, thepharmaceutical composition of the invention will be administrated usingthe appropriate equipments, apparatus, and devices which are known bythe skilled person in art in a therapeutically or prophylacticallyeffective amount, together with a suitable pharmaceutically acceptablevehicle.

Example 5 3D Platform—Encapsulated Stem-Cells

In this example adipose-derived stem cells are encapsulated in athree-dimensional gel to form a 3D platform prior to introduction into apatient. Treatment with encapsulated stem cells provides significantbiologic advantage over treatment with a dispersed suspension of stemcells. For example, the stem cells remain in groups, keeping oninteraction, normal proliferation and gross factor secretion whereas insuspension, the single stem cells are unable to sustain normaldevelopment in form of single cells in suspension. Additionally, thematrix of the 3D platform defends the encapsulated stem cells againstenvironmental changes and mechanical stress upon delivery of the stemcells into the hosting tissue. The matrix of the 3D platform supportsthe stem cells assuring normal metabolism. Advantageously, in thepresent example, the initial amount of available and injected cells isknown to patient and physician, which allows development ofdose-dependent controlled treatment.

Preparation of encapsulated stem cells requires three general steps.First, stem cells must be extracted, and isolated. The extraction andisolation of stem cells can be performed using conventional methodsand/or the methods described herein. Second, the stem cells are mixedwith a liquid phase biocompatible pro-polymer. Third, the mixture iscaused to gel by crosslinking of the pro-polymer to form a polymer. Asresult, the stem cells are embedded in polymeric biodegradable hydrogelnetwork which serves as 3D culture and support system for the stemcells.

In one embodiment, stem cells are derived from adipose tissue asfollows. Adipose tissue is collected from a patient by mini liposuctioninto a sterile syringe in amount of 50-100 ml in a doctor's office. Theextracted adipose tissue in the sterile syringes is processed on site ortransported to a nearby facility for processing. The contents of thesterile syringes is then released into sterile 50 ml tubes and spun in acentrifuge at 200 g for 5 min. After centrifugation, the fraction ofwhite fat is removed. The cell fraction is weighed and Liberase (Roche)is added in final concentration 12 mg/ml (0.28 Wunsch/ml). The cellfraction is digested with Liberase for 30 minutes in 36.6° C. in hot airshaker. At the end of 30 minutes the digestion is halted by addition ofDMEM medium supplemented 15% human plasmanate (commercial). The treatedcell fraction is the washed twice with reconstituted “StemPro”—anon-animal source recombinant medium (Invitrogen). The pellet containingthe cell fraction is then plated in 75 cm² flask and incubated overnightin 5.5% CO2 incubator at 36.6° C. After overnight incubation cells areremoved by exposition to “TrypLE Select”—recombinant trypsin(Invitrogen) for 8 min followed by washing in reconstituted StemPromedium. After washing, the stem cells are resuspended in 1 ml of StemPromedium. The suspension of stem cells can be characterized at this pointby counting the cells using, e.g., a Scepter automated cell counter. A100 μL sample of the suspension is removed for flow cytometry cellcharacterization and microbiology testing.

The remaining 900 μL of the stem cell suspension is used forencapsulation as follows. A 5 ml solution of Fibrinogen is prepared inconcentration 10 mg/ml in tris-based DPBS.2. Stem cells in suspensionare added to the solution of Fibrinogen to achieve final concentrationof 200,000 cells/ml by gentle pipetting. The amount of stem cellsolution to be added can be calculated because the stem cell solutionhas previously been characterized/counted. The mixture of the fibrinogenprecursor and stem cells is the pipetted into 10 ml of thrombin solutionin concentration 50 mg/ml. The resulting mixture is incubated in 5.5%CO2 at 36.6° C. for thirty minutes to form a gel including theencapsulated stem cells.

In a preferred embodiment the gel is formed into microbeads by manual orusing an automatic chip device. The microbeads are preferably 10-50 μLin size. The microbeads are designed and the stem cell concentratedselected such the microbeads can support and maintain the stem cells invivo for a period of 3 to 14 days during which period the stem cellsremain within the microbead and release therapeutic factors into thesurrounding tissues. The microbeads are kept in incubator until 2 hrsbefore scheduled procedure. At that point the product is delivered toSurgical Center at ambient temperature. Alternatively, the encapsulatedstem cells are frozen and delivered in ready-to-use condition.Microbeads can be frozen by slow freezing or vitrification method anddelivered in ready-to-use condition. Alternatively, the stem cells canbe extracted and the 3-D platform and microbeads can be created at thesite of the procedure using appropriate devices.

Example 6 Treatment of Osteoarthritis Utilizing 3D Platform

In this example, treatment of osteoarthritis is performed by injectingencapsulated stem cells in the form of the 3D platform. The 3D platformis created as described above utilizing a fibrin gel encapsulatingadipose-derived autologous stem cells. (Note that in alternativeembodiments stem cells of other types- and origin can be utilized). The3D platform microbeads incorporating the stem cells are then injectedsub-chondrally adjacent damaged tissues such as damaged cartilage of anaffected knee or elbow joint.

The administration of the 3D platform can be carried out by conventionalmeans. In a particular embodiment, the 3D platform is introducedsub-chondrally using devices such as syringes, catheters, trocars,cannulae, etc. The administration can be performedpercutaneously/arthroscopically and can be repeated as necessary toachieve the desired therapeutic effects. For example, the 3D platform isintroduced sub-chondrally using an image-guided needle or similartechnology. In any case, the pharmaceutical composition of the inventionwill be administrated using the appropriate equipments, apparatus, anddevices which are known by the skilled person in art in atherapeutically or prophylactically effective amount.

The microbeads maintain the stem cells in the target location inundifferentiated from and protect the stem cells from chemical andmechanical stress at the site of introduction. The encapsulated stemcells are able to survive and/or proliferate in vivo within themicrobeads for an extended period as compared to stem cells insuspension introduced into the joint. In a preferred embodiment theencapsulated stem cells are maintained for 3-14 days. The encapsulatedstem cells release cytokines, growth factors, anti-inflammatory factorswhich migrate out of the 3D platform into the surrounding tissues. Thesefactors engender a therapeutic effect in the target tissues adjacent thesite of injection of the 3D platform. The cellular factors released fromthe 3D platform are able to migrate into the osteo-arthritic tissues ofthe joint rejuvenating the damaged tissues and thereby stimulating thosetissues to repair themselves and achieve a therapeutic result. At theend of the 3-14 days the fibrin of the microbeads is digested and thestem cells are no longer maintained. The procedure can be repeated asnecessary to achieve the desired therapeutic effect.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. For purposes of summarizing thepresent invention, certain aspects, advantages and novel features of thepresent invention have been described herein. Of course, it is to beunderstood that not necessarily all such aspects, advantages or featureswill be embodied in any particular embodiment of the present invention.Additional advantages and aspects of the present invention are apparentin the following detailed description and claims.

A number of publications and patents have been cited hereinabove. Eachof the cited publications and patents are hereby incorporated byreference in their entireties. All publications, patents and patentapplications are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety.

The foregoing description of preferred embodiments of the presentinvention has been provided for the purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise forms disclosed. Many embodiments were chosenand described in order to best explain the principles of the inventionand its practical application, thereby enabling others skilled in theart to understand the invention for various embodiments and with variousmodifications that are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claims andtheir equivalents.

1. A therapeutic composition for treatment of a patient comprising: aplurality of microbeads of a biocompatible hydrogel; each microbeadcontaining a plurality of stem cells encapsulated within the hydrogel.2. The therapeutic composition of claim 1, wherein the microbeads have avolume between 10 and 50 μL.
 3. The therapeutic composition of claim 1,wherein the microbeads comprise about 200 stem cells per microliter. 4.The therapeutic composition of claim 1, wherein the biocompatiblehydrogel is biodegradable.
 5. The therapeutic composition of claim 1,wherein the biocompatible hydrogel comprises fibrin.
 6. The therapeuticcomposition of claim 1, wherein the microbeads are adapted to protectthe stem cells from mechanical and chemical stress when introduced tothe patient.
 7. The therapeutic composition of claim 1, wherein themicrobeads are adapted to protect the stem cells from mechanical andchemical stress when introduced to the patient.
 8. The therapeuticcomposition of claim 1, wherein the microbeads are adapted to releasecellular factors from said stem cells to a target tissue of the patientinto which said microbeads are introduced.
 9. The therapeuticcomposition of claim 1, wherein the microbeads are adapted to maintainsaid stem cells for a period of three to fourteen days afterintroduction into a target tissue of the patient thereby promotingrelease of cellular factors from said stem cells to said target tissueinto which said microbeads are introduced.
 10. The therapeuticcomposition of claim 1, wherein the microbeads are adapted to maintainsaid stem cells in an undifferentiated condition.
 11. The therapeuticcomposition of claim 1, wherein the stem cells are adipose-derived stemcells.
 12. The therapeutic composition of claim 1, wherein the stemcells are autologous stem cells.
 13. The therapeutic composition ofclaim 1, wherein the stem cells are allogeneic stem cells.
 14. A methodfor making a therapeutic composition for treating a patient, the methodcomprising: (a) extracting adipose tissue; (b) preparing a cellularfraction of said adipose tissue wherein said cellular fraction includesa plurality of stem cells; (c) adding said cellular fraction to agel-forming solution; (d) causing said gel-forming solution to form abiocompatible hydrogel encapsulating said plurality of stem cells; and(e) forming the hydrogel into a plurality of microbeads.
 15. The methodof claim 14, wherein the microbeads have a volume between 10 and 50 μL.16. The method of claim 14, wherein the microbeads comprise about 200stem cells per microliter.
 17. The method of claim 14, wherein thebiocompatible hydrogel comprises fibrin.
 18. The method of claim 14,wherein the stem cells are autologous stem cells.
 19. The method ofclaim 14, wherein the stem cells are allogeneic stem cells.
 20. A methodfor treating a patient comprising: (a) receiving a therapeuticcomposition which includes a plurality of microbeads of a biocompatiblehydrogen wherein each microbead contains a plurality of stem cellsencapsulated within the hydrogel; (b) introducing said therapeuticcomposition adjacent a diseased or injured tissue; and (c) causing therelease of therapeutic cellular factors from said stem cellsencapsulated within the hydrogel to said diseased or injured tissue.